1. Field of the Invention
The present invention relates to a driving mechanism of a mirror applied to a scanning laser microscope, and a spectroscope and a scanning laser microscope comprising a mirror which is driven by the driving mechanism.
2. Description of the Related Art
The following device is disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2000-56233. This device comprises at least one dispersive device for separating the irradiated light wavelength and at least one element arranged in the irradiated light wavelength separation part which re-reflects at least one wavelength band as a target in the direction of the microscope irradiation, i.e., at least partially reflects it. As a result, the wavelength or the wavelength band on the irradiation optical path of the microscope, especially the confocal microscope is focused under the adjustment.
FIG. 1 is a figure which shows a configuration of the above-mentioned confocal scanning laser microscope. The light beam output from the fiber F is shaped to the parallel light beam with the lens K, and is incident to the dispersive device DP. The dispersive device DP separates the light beam into light beams with each wavelength included in the laser beam. The light beam separated by the dispersive device DP, for instance, λ1, λ2, and λ3 reaches the element ST. The element ST has small mirrors S1, S2, and S3 arranged at a position where light beams λ1, λ2, and λ3 are condensed, and other parts thereof are transparent.
The irradiation beam is apart from the center of the dispersive device DP and is led to the upper side. Therefore, the each of partial light beams λ1, λ2, and λ3 enters the upper side of the lens L1, travels a little downward from there and are condensed on the small mirrors S1, S2, and S3. The optical path of the light beam reflected with the small mirrors S1, S2, and S3 inclines further downward, and returns below the element DP by passing the lens L1.
The return light beam reflected with the small mirrors S1, S2, and S3, are condensed after passing the dispersive device DP, and pass the lens LS as a light beam with a plurality of wavelengths. And, the condensed light beam is led to the microscope by passing the pinhole PH and reaches the object by passing the microscope objective as shown in FIG. 2.
Fluorescence from the object reaches each middle area of the small mirrors S1, S2, . . . and passes the light-transparent part ST of the small mirrors S1, S2, . . . as shown in FIG. 3. The wedge prisms GK (GK1, GK2, GK3, and GK4, here) are arranged corresponding to each evaluation optical path passing the light-transparent part ST. It becomes possible to limit the wavelength band by being possible to move the wedge prisms GK along two axes. The fluorescence passing the wedge prisms GK is detected with the optical detectors arranged on each evaluation optical path.
As mentioned above, the device described in the Jpn. Pat. Appln. KOKAI Publication No. 2000-56233 is configured as follows based on the scanning confocal laser microscope. That is, this comprises at least one dispersive device for separating the irradiated light wavelength and at least one element arranged in the irradiated light wavelength separation part which re-reflects at least one wavelength band as a target in the direction of the microscope irradiation, i.e., at least partially reflects it, and it can be condensed in a state that the wavelength or the wavelength is adjusted on the irradiation optical path. As a result, an optical filter (dichroic mirror to separate the excitation light beam and fluorescence) for separating the wavelength is not needed. The degree of the freedom becomes high.
However, the beam quality of the laser beam is gradually degraded by passing or reflecting the optical system. The degradation happens one-sidedly, and is not recovered. The diffraction spot broadens since the condense performance in which the objective originally has cannot be obtained when the beam quality degrades. Therefore, both of the in-plane resolution and the confocal effect as the confocal laser microscope degrade. In a word, the performance of the confocal laser microscope greatly depends on especially the optical accuracy on the illumination optical path.
The beam quality degradation of the laser beam is often caused by especially surface accuracy defective of the mirror. The surface accuracy below {fraction (1/10)}λ is requested for the mirror usually. Since the laser beam is condensed and reflected to the small mirrors S1, S2, . . . , in the Jpn. Pat. Appln. KOKAI Publication No. 2000-56233, higher surface accuracy is requested. In addition, the dust which adheres to the mirror surface affects an important influence on the degradation of the beam quality.
This dust will be considered. There is a possibility that the dust which exists in the natural world and floats in the device adheres to the mirror surface. Additionally, there is also a possibility that the laser beam collects the dust which floats in the device since the effect of an optical trap is caused at the condensed position when the laser beam is condensed. In addition, the fluorescent tenebrescence method is widely used as an application which uses the fluorescent protein in recent years. This technique is a method of not only detecting fluorescence from the sample by irradiating the laser beam to the sample but also positively discoloring the fluorescent materials which is used as the sample to observe the recovery of the fluorescent material. Therefore, stronger laser power is requested compared with a usual fluorescent observation. Therefore, the possibility of the collection of the dust by the above-mentioned optical trap becomes larger.
The laser microscope to observe two-photon fluorescence from the sample attracts especially attention in recent years. The strong laser power with from several mill watts to several tens mill watts is requested to generate two photon fluorescence from the sample. Therefore, the possibility of the collection of the dust by an optical trap extremely becomes large in this laser microscope.
To satisfy the above-mentioned application by the configuration according to the Jpn. Pat. Appln. KOKAI Publication No. 2000-56233 and provide the device to observe two-photon fluorescence, it is necessary to exclude the dust in the device with an extremely high level. Therefore, an important influence is affected on the cost on the manufacture.
The configuration according to the Jpn. Pat. Appln. KOKAI Publication No. 2000-56233 has a problem that the freedom degree of the arrangement of an optical detector is low and is limited.
In the configuration according to the Jpn. Pat. Appln. KOKAI Publication No. 2000-56233, the light from the sample reaches each middle area of the small mirrors S1, S2, . . . , and passes the light-transparent part ST of the small mirrors S1, S2, . . . as shown in FIG. 3. The wedge prisms GK (GK1, GK2, GK3, and GK4, here) are arranged corresponding to each evaluation optical path passing the light-transparent part ST. Each light flux is bent by the dispersion with the wedge prisms GK, and, in addition, is condensed by the lens KO.
However, the light flux which does not pass the wedge prisms GK goes toward a direction of an optical detector at the different angle as shown in FIG. 4. Even when the wedge angle of the prism becomes large and the angle difference is about 60 degrees at most. Though the refraction angle becomes large if the wedge angle of the wedge prism is large, the wavelength decomposition accuracy lowers since the distance from the condensed position to an incident position to the wedge prism increases. In a word, the angle difference and the wavelength decomposition accuracy have the conflicting relations.
Therefore, since the layout is limited to especially in a case of using a photo-multiplier of a side on type as an optical detector, it is necessary to keep away the position of an optical detector from the element ST which holds the mirror, and, as a result, the device is enlarged. This is an unavoidable whenever the above-mentioned conventional configuration such that a desired spectrum band is limited by penetrating the light from the sample at the position of the element ST which holds the mirror, and refracting it with the prism.
In addition, the independent confocal diaphragm is not arranged on the separated each optical detection path and it is difficult to be arranged according to the configuration of Jpn. Pat. Appln. KOKAI Publication No. 2000-56233.
Generally, it is ordinary that fluorescence from the sample is slight as the photo-multipliers are needed as an optical detector. Since tenebrescence of a fluorescent sample becomes remarkable when the excited light (laser light) is strong, it is general to try to lower the excited light amount as much as possible within the permissible range while the observer considers the balance of the noise of the tenebrescence of the sample and the obtained image. Therefore, it becomes an extremely important to suppress the loss of the fluorescence as much as possible in this kind of device.
The sample labeled by two fluorescence dyes (DAPI, CY5) is considered.
The DAPI has the absorption wavelength band in the UV band (340 to 365 nm). The peak of the fluorescent wavelength emitted from the DAPI is about 450 nm. On the other hand, the CY5 has the absorption wavelength band in red band (630 to 650 nm), and the peak of the fluorescent wavelength is about 670 nm.
Here, the spot formed by the fluorescence at the image formation position will be described referring to FIG. 2.
When the laser spot is formed with the objective 21 on the sample 22, the fluorescence emitted from here focuses the spot at the image formation position where the confocal diaphragm 16 is arranged, that is, the image formation position by the lens 23 through the objective 21, the image formation lens 20, and the pupil projection lens 19. The spot diameter (diffraction diameter) is shown by the following expression.φ=1.22×λ/NA
NA is an opening of the lens 23, and λ is wavelength.
The spot diameter of the CY5 (fluorescent wavelength 670 nm) differs from by the DAPI (fluorescent wavelength 450 nm) about 1.5 times and is larger than that from this expression.
Therefore, to secure the confocal effect, the diameter of the confocal diaphragm 16 is set according to the spot diameter of the DAPI in the above-mentioned conventional technology. This set value becomes optimal for the DAPI. However, since the confocal diaphragm 16 needlessly narrows this set value, the valuable fluorescence is lost. There is a defect that when the diameter of the confocal diaphragm 16 is matched to the CY5, an enough confocal effect cannot be achieved by the DAPI. Thus, since the confocal diaphragm is not arranged on each detection optical path, there is the above-mentioned defect.
At least one wedge prism which disperses the fluorescence in a direction different from the direction where the fluorescence from the sample is dispersed to each detection optical path exists.
Therefore, it is realistically difficult to arrange the confocal diaphragm on the optical paths, respectively. The reason is why it is necessary to re-arrange and move the prism for the light flux to focus it by canceling dispersion in synchronous with the movement of the above-mentioned wedge prism.
In addition, in the above-mentioned conventional configuration, in order to apply to several kinds of laser beams, it is necessary to change and adjust the small mirror to reflect the laser beam.
As mentioned above, the laser beam, that is, the light beam separated into the wavelength included in the laser beam in the dispersive device DP has the small mirrors S1, S2, and S3 in the condensing positions, and other parts thereof reach the element ST which is a transparent mirror holding body. In a word, the small mirror is arranged always, for instance, as the mirror holding glass stick at a position where the laser beam is condensed corresponding to the wavelength.
Since the stick excitation selector (mirror holding body ST with small mirrors S1, S2, S3, . . . ) can be exchanged, the single or a plurality of wavelengths can be arbitrarily configured.
The conventional example, the laser beam which is the excitation light beam is reflected, the small mirrors S1, S2, S3, . . . are provided to lead the light from the sample which contains the fluorescence to the optical detector, and the element ST which is used as the transparent mirror holding body is required as other parts. It is possible to apply to the excitation light beam in single wavelength or a plurality of wavelength bands by the mirror holding body ST. However, it is necessary to exchange the holding body ST or the small mirror in every case for the laser beam from which various combinations are considered. However, since high accuracy is required for the angle of the small mirror and the position of the mirror to the holding body ST in order to reflect and lead the laser beam surely with a small mirror to the microscope, a difficult adjustment to secure accuracy when exchanging is needed.
The conventional configuration has a problem that it is difficult to detect the fluorescence by the best condition. For instance, a case that the ST comprising a small mirror in a spectrum band where the excitation wavelength correspond to 488, 543, and 633 nm is used and the fluorescence of the labeled sample is observed with two fluorescence dyes (FITC, CY5) is considered. The wavelengths of 488 and 633 nm are used for the excitation wavelength. Therefore, the wavelength of 543 nm is not used.
At this time, the fluorescence emitted from the labeled part with the FITC contains the wavelength of 543 nm. However, the light beam of the wavelength of 543 nm cannot be detected as the fluorescence from the sample since it is reflected with the small mirror. Therefore, it is necessary to arrange a lot of small mirrors to correspond to a lot of laser beams by one mirror holding body ST. However, though it becomes possible to apply a lot of laser beams by the above-mentioned method, the fluorescence included in the light from the sample having the same wavelength as the wavelength of the laser beam not used as excitation light beam is always reflected.
Besides the above-mentioned conventional technology, in the International Patent Application KOHYO Publication No. 9-502269, the spectroscope in which the fluorescent light flux is dispersed according to the spectrum decomposition means such as prisms, the first spectrum range is narrowed at one side, on the other hand, the second range by reflecting at least part of the spectrum range where the diaphragm is not passed to configure two optical paths which configures the second spectrum range and the optical detector is provided to each optical path.
The outline configuration of spectroscope 216 of International Patent Application KOHYO Publication No. 9-502269 will be explained referring to FIG. 5. FIG. 5 is a figure which shows the configuration when three optical detectors are provided.
The selection device 225 has the spectrum decomposition means 227 for separating the light flux 214 and means 228 for narrowing the first spectrum range 229 at one side and reflecting at least part 230 of the spectrum range which does not pass the diaphragm on the other hand. The optical detector 226 has the first optical detector 231 arranged on the optical path within the narrowed first spectrum range 229 and the second optical detector 232 arranged on the optical path within the range of the reflected spectrum. The selection device 225 is arranged on the optical path within reflecting range 230 of the spectrum and has means 233 for narrowing the second spectrum range 234. The second optical detector 232 is arranged on the optical path within the narrowed second spectrum range 234. The third optical detector 236 is arranged on the optical path within the further reflected spectrum range 235. Means 238 for narrowing the third spectrum range 237 on the optical path within the reflected spectrum range is arranged. The third optical detector 236 is arranged on the optical path within the narrowed third spectrum range 237.
It becomes possible to select the three spectrum ranges 229, 234, and 237 as a total and perform optical detection as mentioned above.
The configuration of the device comprising three optical detectors is shown above. When the number of detectors is two, the third optical detector 236 has been similarly omitted in that case.
In the configuration of the above-mentioned International Patent Application KOHYO Publication No. 9-502269, the light flux 214 is separated in the spectrum by the spectrum decomposition means 227. The first spectrum range 229 is narrowed in one side, and is divided into the wavelength bands of each of fluorescence with means 228 (changeable slit) which reflects at least part 230 within the spectrum range which does not pass the diaphragm on the other hand reflects. However, since the light flux diameter of light flux 214 has some size, it is not divided into each detection optical path with the enough spectrum resolution. Though the distance of the spectrum decomposition means 227 and reflection means 228 may be taken long to raise the spectrum resolution, since the device becomes large, it is not realistic.
The technology which arranges the condenser lens behind the spectrum decomposition means is known as means to make up for the fault that the spectrum accuracy is limited by the size of the light flux diameter. As a result, the spectrum resolution is improved more than the configurations without the condenser lens shown in FIG. 5, since light flux can be narrowed to the diffraction diameter.
However, the light flux is condensed by the condenser lens, the condensed position cannot be set at the best position for both narrowing means 228 and 233, for instance in FIG. 5. The will be explained to be assumed to set the condensed position in narrowing means 228. In this case, though the spectrum of extremely high accuracy is possible since light flux is narrowed even to the diffraction diameter in narrowing means 228, an optical position of the narrowing means 233 largely shifts from the condensed position. Therefore, since the light flux broadens at this position, the spectrum of high accuracy cannot be achieved.
Therefore, when the same spectrum accuracy is obtained, the condensed position will be set at the middle position of the narrowing means 228 and 233. However, since this position shifts from the condensed position for each means, ideal spectrum accuracy cannot be achieved.